Cytochrome oxidase is an important structural and functional constituent of the mitochondrial inner membrane. As such it has become a model enzyme for studies on the mechanism of electron transport and energy coupling. In addition, it is well suited to probe questions related to the biosynthesis and assembly of integral membrane proteins. In yeast, cytochrome oxidase is composed of seven different subunit polypeptides. The three largest subunits are synthesized internally by the mitochondrial translational system. The four smaller subunits whose functions are still unclear, are known to be products of cytoribosomal protein synthesis. The dual origin of the enzyme is also reflected in the location of the genes coding for the structural components. Genetic and physical analyses of yeast mitochondrial DNA have revealed that the internally synthesized subunits are also encoded in the organellar genome. Their genes have been sequenced and precisely mapped on the genome. Despite the significant recent advances in defining the contribution of the mitochondrial genetic system, many important aspects of the biosynthesis of this enzyme remain unclear. Very little information exists concerning the structural genes and regulatory elements in nuclear DNA that influence the expression of cytochrome oxidase. The present application encompasses two general areas of studies. The first will attempt to exploit a recently acquired collection of nuclear respiratory deficient mutants of yeast to identify the set of genes required for the expression of cytochrome oxidase. The genes coding for the structural subunits will be cloned and their sequences examined for possible common regulatory features. Secondly, mitochondrial and nuclear mutants defective in cytochrome oxidase will be used to study the sequence of events leading to the formation of the functional holoenzyme.